In this application the interferon nomenclature announced in Nature, 286, p. 2421 (Jul. 10, 1980) will be used. This nomenclature replaces that used in our earlier applications from which this application claims priority. E.g., IF is now designated IFN and fibroblast interferon is now designated IFN-β.
Two classes of interferons (“IFN”) are known to exist. Interferons of Class I are small, acid stable (glyco)-proteins that render cells resistant to viral infection (A. Isaacs and J. Lindenmann, “Virus Interference I. The Interferon”, Proc. Royal Soc. Ser. B., 147, pp. 258-67 (1957) and W. E. Stewart, II, The Interferon System, Springer-Verlag (1979) (hereinafter “The Interferon System”)). Class II IFNs are acid labile. At present, they are poorly characterized. Although to some extent cell specific (The Interferon System, pp. 135-45), IFNs are not virus specific. Instead, IFNs protect cells against a wide spectrum of viruses.
Human interferon (“HuIFN”) has been classified into three groups α, β and γ. HuIFN-β or fibroblast interferon is produced upon appropriate induction in diploid fibroblast cells. It is also produced in minor amounts, together with a major amount of HuIFN-α, in lymphoblastoid cells. IFN-β made from these cells has been extensively purified and characterized (E. Knight, Jr., “Interferon: Purification And Initial Characterization From Human Diploid Cells”, Proc. Natl. Acad. Sci. USA, 73, pp. 520-23 (1976)). It is a glyco-protein of about 20,000 molecular weight (M. Wiranowska-Stewart, et al., “Contributions Of Carbohydrate Moieties To The Physical And Biological Properties Of Human Leukocyte, Lymphoblastoid And Fibroblast Interferons”, Abst. Ann. Meeting Amer. Soc. Microbiol., p. 246 (1978)). It is also heterogeneous in regard to size presumably because of the carbohydrate moities.
The amino acid composition of authentic human fibroblast interferon has also been reported (E. Knight, Jr., et al., “Human Fibroblast Interferon: Amino Acid Analysis And Amino-Terminal Amino Acid Sequence”, Science, 207, pp. 525-26 (1980)). And, elucidation of the amino acid sequence of authentic human fibroblast interferon is in progress. To date, the amino acid sequence of the NH2 terminus of the authentic mature protein has been reported for the first 13 amino acid residues: Met-Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser . . . (E. Knight, Jr., et al., supra).
Two distinct genes, one located on chromosome 2, the other on chromosome 5, have been reported to code for IFN-β (D. L. Slate and F. H. Ruddle, “Fibroblast Interferon In Man Is Coded By Two Loci On Separate Chromosomes”, Cell, 16, pp. 171-80 (1979)). Other studies, however, indicate that the gene for IFN-β is located on chromosome 9 (A. Medger, et al., “Involvement Of A Gene On Chromosome 9 In Human Fibroblast Interferon Production”, Nature, 280, pp. 493-95 (1979)).
Although authentic HuIFN-β is glycosylated, removal of the carbohydrate moiety (P. J. Bridgen, et al., “Human Lymphoblastoid Interferon”, J. Biol. Chem., 252, pp. 6585-87 (1977)) or synthesis of IFN-β in the presence of inhibitors which purport to preclude glycosylation (W. E. Stewart, II, et al., “Effect of Glycosylation Inhibitors On The Production And Properties Of Human Leukocyte Interferon”, Virology, 97, pp. 473-76 (1979); J. Fujisawa, et al., “Nonglycosylated Mouse L Cell Interferon Produced By The Action Of Tunicamycin”, J. Biol. Chem., 253, pp. 8677-79 (1978); E. A. Havell, et al., “Altered Molecular Species Of Human Interferon Produced In The Presence Of Inhibitors of Glycosylation”, J. Biol. Chem., 252, pp. 4425-27 (1977); The Interferon System, p. 181) yields a smaller form of IFN-β which still retains most or all of its IFN activity.
HuIFN-β, like many human proteins, may also be polymorphic. Therefore, cells of particular individuals may produce IFN-β species within the more general IFN-β class which are physiologically similar but structurally slightly different from the prototype of the class of which it is a part. Therefore, while the protein structure of the IFN-β may be generally well-defined, particular individuals may produce IFN-βs that are slight variations thereof.
IFN-β is usually not detectable in normal or healthy cells (The Interferon System, pp. 55-57). Instead, the protein is produced as a result of the cell's exposure to an IFN inducer. IFN inducers are usually viruses but may also be non-viral in character, such as natural or synthetic double-stranded RNA, intracellular microbes, microbial products and various chemical agents. Numerous attempts have been made to take advantage of these non-viral inducers to render human cells resistant to viral infection (S. Baron and F. Dianzani (eds.), Texas Reports On Biology And Medicine, 35 (“Texas Reports”), pp. 528-40 (1977)). These attempts have not been very successful. Instead, use of exogenous HuIFN-β itself is now preferred.
Interferon therapy against viruses and tumors or cancers has been conducted at varying dosage regimes and under several modes of administration (The Interferon System, pp. 305-321). For example, interferon has been effectively administered orally, by innoculation—intravenous, intramuscular, intranasal, intradermal and subcutaneous—, and in the form of eye drops, ointments and sprays. It is usually administered one to three times daily in dosages of 104 to 107 units. The extent of the therapy depends on the patient and the condition being treated. For example, virus infections are usually treated by daily or twice daily doses over several days to two weeks and tumors and cancers are usually treated by daily or multiple daily doses over several months or years. The most effective therapy for a given patient must of course be determined by the attending physician, who will consider such well known factors as the course of the disease, previous therapy, and the patient's response to interferon in selecting a mode of administration and a dosage regime.
As an antiviral agent, HuIFN has been used to treat the following: respiratory infections (Texas Reports, pp. 486-96); herpes simplex keratitis (Texas Reports, pp. 497-500; R. Sundmacher, “Exogenous Interferon in Eye Diseases”, International Virology IV, The Hague, Abstract nr. W2/11, p. 99 (1978)); acute hemorrhagic conjunctivitis (Texas Reports, pp. 501-10); adenovirus keratoconjunctivitis (A. Romano, et al., ISM Memo I-A8131 (October, 1979)); varicella zoster (Texas Reports, pp. 511-15); cytomegalovirus infection (Texas Reports, pp. 523-27); and hepatitis B (Texas Reports, pp. 516-22). See also The Interferon System, pp. 307-19. However, large-scale use of IFN as an antiviral agent requires larger amounts of IFN than heretofore have been available.
IFN has other effects in addition to its antiviral action. For example, it antagonizes the effect of colony stimulating factor, inhibits the growth of hemopoietic colony-forming cells and interferes with the normal differentiation of granulocyte and macrophage precursors (Texas Reports, pp. 343-49). It also inhibits erythroid differentiation in DMSO-treated Friend leukemia cells (Texas Reports, pp. 420-28). It is significant that some cell lines may be considerably more sensitive to HuIFN-β than to HuIFN-α in these regards (S. Einhorn and H. Strander, “Is Interferon Tissue-Specific?—Effect Of Human Leukocyte And Fibroblast Interferons On The Growth Of Lymphoblastoid And Osteosarcoma Cell Lines”, J. Gen. Virol., 35, pp. 573-77 (1977); T. Kuwata, et al., “Comparison Of The Suppression Of Cell And Virus Growth In Transformed Human Cells By Leukocyte And Fibroblast Interferon”, J. Gen. Virol., 43, pp. 435-39 (1979)).
IFN may also play a role in regulation of the immune response. For example, depending upon the dose and time of application in relation to antigen, IFN can be both immunopotentiating and immunosuppressive in vivo and in vitro (Texas Reports, pp. 357-69). In addition, specifically sensitized lymphocytes have been observed to produce IFN after contact with antigen. Such antigen-induced IFN could therefore be a regulator of the immune response, affecting both circulating antigen levels and expression of cellular immunity (Texas Reports, pp. 370-74). IFN is also known to enhance the activity of killer lymphocytes and antibody-dependent cell-mediated cytotoxicity (R. R. Herberman, et al., “Augmentation By Interferon Of Human Natural And Antibody-Dependent Cell-Mediated Cytotoxicity”, Nature, 277, pp. 221-23 (1979); P. Beverley and D. Knight, “Killing Comes Naturally”, Nature, 278, pp. 119-20 (1979); Texas Reports, pp. 375-80; J. R. Huddlestone, et al., “Induction And Kinetics Of Natural Killer Cells in Humans Following Interferon Therapy”, Nature, 282, pp. 417-19 (1979); S. Einhorn, et al., “Interferon And Spontaneous Cytotoxicity In Man. II. Studies In Patients Receiving Exogenous Leukocyte Interferon”, Acta Med. Scand., 204, pp. 477-83 (1978)). Both may be directly or indirectly involved in the immunological attack on tumor cells.
Therefore, in addition to its use as an antiviral agent, HuIFN has potential application in antitumor and anticancer therapy (The Interferon System, pp. 319-21 and 394-99). It is now known that IFNs affect the growth of many classes of tumors in many animals (The Interferon System, pp. 292-304). They, like other anti-tumor agents, seem most effective when directed against small tumors. The antitumor effects of animal IFN are dependent on dosage and time but have been demonstrated at concentrations below toxic levels. Accordingly, numerous investigations and clinical trials have been and continue to be conducted into the antitumor and anticancer properties of HuIFNs. These include treatment of several malignant diseases such as osteosarcoma, acute myeloid leukemia, multiple myeloma and Hodgkin's disease (Texas Reports, pp. 429-35). In addition, HuIFN-β has recently been shown to cause local tumor regression when injected into subcutaneous tumoral nodules in melanoma and breast carcinoma-affected patients (T. Nemoto, et al., “Human Interferons And Intralesional Therapy Of Melanoma And Breast Carcinoma”, Amer. Assoc. For Cancer Research, Abs nr. 993, p. 246 (1979)). Although the results of these clinical tests are encouraging, the antitumor and anticancer applications of IFN-β have been severely hampered by lack of an adequate supply of purified IFN-β.
Significantly some cell lines which resist the anticellular effects of IFN-α remain sensitive to IFN-β. This differential effect suggests that IFN-β may be usefully employed against certain classes of resistant tumor cells which appear under selective pressure in patients treated with high doses of IFN-α (T. Kuwata, et al., supra; A. A. Creasy, et al., “The Role of G0-G1 Arrest In The Inhibition Of Tumor Cell Growth By Interferon”, Abstracts, Conference On Regulatory Functions Of Interferons, N.Y. Acad. Sci., nr. 17 (Oct. 23-26, 1979)).
At the biochemical level IFNs induce the formation of at least 3 proteins, a protein kinase (B. Lebleu, et al., “Interferon, Double-Stranded RNA And Protein Phosphorylation”, Proc. Natl. Acad. Sci. USA, 73, pp. 3107-11 (1976); A. G. Hovanessian and I. M. Kerr, “The (2′-5′) Oligoadenylate (ppp A2′-5A2′-5′A) Synthetase And Protein Kinase(s) From Interferon-Treated Cells”, Eur. J. Biochem., 93, pp. 515-26 (1979)), a (2′-5′)oligo(A) polymerase (A. C. Hovanessian, et al., “Synthesis Of Low-Molecular Weight Inhibitor Of Protein Synthesis With Enzyme From Interferon-Treated Cells”, Nature, 268, pp. 537-39 (1977); A. G. Hovanessian and I. M. Kerr, Eur. J. Biochem, supra) and a phosphodiesterase (A. Schmidt, et al., “An Interferon-Induced Phosphodiesterase Degrading (2′-5′)oligoisoadenylate And The C-C-A Terminus Of tRNA”, Proc. Natl. Acad. Sci. USA, 76, pp. 4788-92 (1979)).
Both IFN-β and IFN-α appear to trigger similar enzymatic pathways (C. Baglioni, “Interferon-Induced Enzymatic Activities And Their Role In The Antiviral State”, Cell, 17, pp. 255-64 (1979)) and both may share a common active core because they both recognize a chromosome 21-coded cell receptor (M. Wiranowska-Stewart, “The Role Of Human Chromosome 21 In Sensitivity To Interferons”, J. Gen. Virol., 37, pp. 629-34 (1977)). The appearance of one or more of these enzymes in cells treated with IFN should allow a further characterization of proteins with IFN-like activity.
Today, HuIFN-β is produced by human cell lines grown in tissue culture. It is a low yield, expensive process. One large producer makes only 40-50×108 units of crude IFN-β per year (V. G. Edy, et al., “Human Interferon: Large Scale Production In Embryo Fibroblast Cultures”, in Human Interferon (W. R. Stinebring and P. J. Chapple, eds.), Plenum Publishing Corp., pp. 55-60 (1978)). On purification by adsorption to controlled pore glass beads, IFN-β of specific activity of about 106 units/mg may be recovered in 50% yield from the crude cell extracts (A. Billiau, et al., “Human Fibroblast Interferon For Clinical Trials: Production, Partial Purification And Characterization”, Antimicrobial Agents And Chemotherapy, pp. 49-55 (1979)). Further purification to a specific activity of about 109 units/mg is accomplished by zinc chelate affinity chromatography in about 100% yield (A. Billiau, et al., “Production, Purification And Properties Of Human Fibroblast Interferon”, Abstracts, Conference On Regulatory Functions Of Interferons, N.Y. Acad. Sci., nr 29 (Oct. 23-26, 1979)). Because the specific activity of HuIFN-β is so high, the amount of IFN-β required for commercial applications is low. For example, 100 g of pure IFN-β would provide between 3 and 30 million doses.
Recent advances in molecular biology have made it possible to introduce the DNA coding for specific non-bacterial eukaryotic proteins into bacterial cells. In general, with DNA other than that prepared via chemical synthesis, the construction of such recombinant DNA molecules comprises the steps of producing a single-stranded DNA copy (cDNA) of a purified messenger RNA (mRNA) template for the desired protein; converting the cDNA to double-stranded DNA; linking the DNA to an appropriate site in an appropriate cloning vehicle to form a recombinant DNA molecule and transforming an appropriate host with that recombinant DNA molecule. Such transformation may permit the host to produce the desired protein. Several non-bacterial genes and proteins have been obtained in E. coli using recombinant DNA technology. These include, for example, IFN-α (S. Nagata, et al., “Synthesis In E. coli Of A Polypeptide With Human Leukocyte Interferon Activity”, Nature, 284, pp. 316-20 (1980)). In addition, recombinant DNA technology has been employed to produce a plasmid said to contain a gene sequence coding for IFN-β (T. Taniguchi, et al., “Construction And Identification of A Bacterial Plasmid Containing The Human Fibroblast Interferon Gene Sequence”, Proc. Japan Acad. Ser. B, 55, pp. 464-69 (1979)).
However, in neither of the foregoing has the actual gene sequence of IFN-β been described and in neither has that sequence been compared to the initial amino acid sequence or amino acid composition of authentic IFN-β. The former work is directed only to IFN-α, a distinct chemical, biological and immunological Class I interferon from IFN-β (cf. supra). The latter report is based solely on hybridization data. These data alone do not enable one to determine if the selected clone contains the complete or actual gene sequence coding for IFN-β or if the cloned gene sequence will be able to express IFN-β in bacteria. Hybridization only establishes that a particular DNA insert is to some extent homologous with and complementary to a mRNA component of the poly(A) RNA that induces interferon activity when injected into oocytes. Moreover, the extent of any homology is dependent on the hybridization conditions chosen for the screening process. Therefore, hybridization to a mRNA component of poly(A) RNA alone does not demonstrate that the selected DNA sequence is a sequence which codes for HuIFN-β or a polypeptide which displays the immunological or biological activity of HuIFN-β or that such sequence will be useful in producing such polypeptides in appropriate hosts.
At a seminar in Zurich on Feb. 25, 1980, Taniguchi stated that he had determined the nucleotide sequence for one of his hybridizing clones. He also stated that the first 13 amino acids coded for by that sequence were identical to that determined by Knight, et al., supra, for authentic HuIFN-β. Taniguchi did not disclose the full nucleotide sequence for his clone or compare its amino acid composition with that determined for authentic HuIFN-β. Taniguichi has since reported the full nucleotide sequence for his hybridizing clone (T. Taniguichi et al., Gene, 10, pp. 11-15 (1980)). The sequence differs by one nucleotide from that described and claimed in British patent application 8011306, filed Apr. 3, 1980, an application to which the present application claims priority. The amino acid sequence reported by Taniguichi is identical to the amino acid sequence described and claimed in the foregoing application 8011306. Taniguichi had also not reported the expression in an appropriate host of polypeptides which display an immunological or biological activity of HuIFN-β at the time of the filing of British patent application 80.18701, filed Jun. 6, 1980, an application to which this application claims priority. It is this expression in a host of polypeptide(s) displaying an immunological or biological activity of HuIFN-β and the methods, polypeptides, genes and recombinant DNA molecules thereof, which characterize this invention.
Nor is this invention addressed as is the apparent suggestion of Research Disclosure No. 18309, pp. 361-62 (1979) to prepare pure or substantially pure IFN-α mRNA before attempting to clone the IFN gene or to produce fibroblast interferon-like polypeptides in bacterial hosts.
Finally, it should be recognized that the selection of a DNA sequence or the construction of a recombinant DNA molecule which hybridizes to a mRNA from polyA RNA, that mRNA producing HuIFN activity in oocytes, is not sufficient to demonstrate that the DNA sequence or the hybrid insert of the recombinant DNA molecule corresponds to HuIFN. Instead, in the absence of a comparison of the amino acid sequence coded for by a particular DNA sequence and the amino acid sequence of the authentic protein, only the production of a polypeptide that displays an immunological or biological activity of HuIFN can actually demonstrate that the selected DNA sequence or constructed recombinant DNA molecule corresponds to HuIFN. More importantly, it is only after such HuIFN activity is shown that the DNA sequence, recombinant DNA molecule or sequences related to them may be usefully employed to select other sequences corresponding to HuIFN in accordance with this invention or to produce recombinant DNA molecules that may express products having an immunological or biological activity of HuIFN-β.
It will therefore be appreciated from the foregoing that the problem of producing HuIFN-β with the use of recombinant DNA technology is much different than any of the above described processes. Here, a particular DNA sequence of unknown structure—that coding for the expression of HuIFN-β in an appropriate host—must be found in and separated from a highly complex mixture of DNA sequences in order for it to be used in the production of HuIFN-β. Furthermore, this location and separation problem is exacerbated by the predicted exceedingly low concentration of the desired DNA sequence in the complex mixture and the lack of an effective means for rapidly analyzing the many DNA sequences of the mixture to select and separate the desired sequence.